HISTONES AND NUCLEOSOMES IN ARCHAEA (ARCHAEBACTERIA)

我们发现古生菌(以前的考古菌)含有 具有初级序列的小DNA结合蛋白，并预测 次级结构，与核小体核心组蛋白相同。这些 古菌组蛋白在体内和体外结合和紧凑DNA 看起来像核小体的结构。这一发现，加上 随着最近认识到所有的基本分子组成 转录起始在古生代和真核生物中保守 (以前的真核生物)暗示，细胞的结构和功能 在基因组压缩和基因表达调控中，核小体 从一个更简单的系统进化而来，这个系统仍然保留在古生代。这个 提出的实验是为了确定这是否正确。我们会 确定HMfA和HMFB，组蛋白A和B的三维结构 费维氏甲烷杆菌及其分子化学计量学和构型 它们形成的核小体样结构(NLS)。蛋白质-DNA 决定这些古生组蛋白在哪里以及如何结合的相互作用 将DNA分子限制在NLS中将由定点确定 诱变及其分离、筛选和鉴定 首选和高亲和力的DNA结合位点。核小体是 在染色质内特别定位和重新定位以调节 真核基因在转录启动水平上的表达，以及 我们将确定这是否也适用于古生菌NLS。我们有 已经确定的条件，可以打开和关闭四个非常强大的古生菌 体内的启动子，以及分离组装和交叉连接的NLS的程序。 在这些不同的条件下原位连接在这些细胞中。我们会 确定NL在体内组装的位置，与下列位置的关系 这些启动子在以下条件下指导转录启动 通过识别核酸酶保护位点来确定启动子的活性和非活性 通过探针杂交、间接末端标记和引物延伸 程序。确定基因表达信号是如何获得的，以及 从染色质内部激活仍然是最重要的 生物医学研究中的问题，也是最难解决的问题之一 试验性的。拟议中的实验不仅将确定 组蛋白、核小体和染色质起源于古生代，但也 这个更简单的系统在多大程度上为研究提供了一个有效的模型 核小体的结构、核小体的定位和核小体的 核小体调节特定基因表达的机制。

We have discovered that the Archaea (formerly Archaebacteria) contain small DNA binding proteins that have primary sequences, and predicted secondary structures, in common with the nucleosome core histones. These archaeal histones bind and compact DNA in vivo and in vitro into structures which visibly resemble nucleosomes. This discovery, coupled with the recent recognition that all the basic molecular components of transcription initiation are conserved in the Archaea and Eukarya (formerly Eukaryotes) suggests that the structure, and the functions of nucleosomes in genome compaction and in regulating gene expression, have evolved from a simpler system that is still retained in the Archaea. The experiments proposed are to determine if this is correct. We will determine the 3D structure of HMfA and HMfB, histones A and B from Methanothermus fervidus, and the molecular stoichiometry and architecture of the nucleosome-like structures (NLS) that they form. The protein-DNA interactions that determine where and how these archaeal histones bind and constrain DNA molecules into NLS will be determined by site-directed mutagenesis and by the isolation, selection and characterization of preferred and high affinity DNA binding sites. Nucleosomes are specifically positioned and re-positioned within chromatin to regulate eukaryotic gene expression at the level of transcription initiation, and we will determine if this is also the case for the archaeal NLS. We have established conditions that turn on and off four very strong archaeal promoters in vivo, and also procedures to isolate NLS assembled and cross- linked in situ in these cells under these different conditions. We will determine where NLS are assembled in vivo, in relationship to the sites at which these promoters direct transcription initiation, under conditions of promoter activity and inactivity, by identifying nuclease protected sites by probe hybridizations, indirect end-labeling and primer extension procedures. Determining how gene expression signals are accessed and activated from within chromatin remains one of the most important questions in biomedical research, and one of the most difficult to address experimentally. The experiments proposed will not only establish if histones, nucleosomes and chromatin originated in the Archaea, but also the extent to which this simpler system provides a valid model for studies of the structure of the nucleosome, of nucleosome positioning, and of the mechanisms by which nucleosomes regulate specific gene expression.